multiresiduemethodforquantificationofsulfonamidesand...

Research ArticleMultiresidue Method for Quantification of Sulfonamides andTrimethoprim in Tilapia Fillet by Liquid ChromatographyCoupled to Quadrupole Time-of-Flight Mass SpectrometryUsing QuEChERS for Sample Preparation

Kátia S. D. Nunes,1 Márcia R. Assalin,2 José H. Vallim,2 Claudio M. Jonsson,2

Sonia C. N. Queiroz,2 and Felix G. R. Reyes 1

1Department of Food Science, School of Food Engineering, University of Campinas, Rua Monteiro Lobato 80,13083-862 Campinas, SP, Brazil2Embrapa Meio Ambiente, P.O. Box 69, 13820-000 Jaguariúna, SP, Brazil

Correspondence should be addressed to Felix G. R. Reyes; [email protected]

Received 25 August 2017; Revised 14 December 2017; Accepted 31 December 2017; Published 1 March 2018

Academic Editor: Gauthier Eppe

Copyright © 2018 Kátia S. D. Nunes et al. .is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

A multiresidue method for detecting and quantifying sulfonamides (sulfapyridine, sulfamerazine, sulfathiazole, sulfamethazine,sulfadimethoxine, sulfamethoxazole, and sulfamethoxypyridazine) and trimethoprim in tilapia fillet (Oreochromis niloticus) usingliquid chromatography coupled to mass spectrometry was developed and validated. .e sample preparation was optimized usingthe QuEChERS approach..e chromatographic separation was performed using a C18 column and 0.1% formic acid in water andacetonitrile as the mobile phase in the isocratic elution mode. Method validation was performed based on the CommissionDecision 2002/657/EC and Brazilian guideline. .e validation parameters evaluated were linearity (r ≥ 0.99); limits of detection(LOD) and quantification (LOQ), 1 ng·g−1 and 5 ng·g−1, respectively; intraday and interdays precision (CV lower than 19.4%)..edecision limit (CCα 102.6–120.0 ng·g−1 and 70 ng·g−1 for sulfonamides and trimethoprim, respectively) and detection capability(CCβ 111.7–140.1 ng·g−1 and 89.9 ng·g−1 for sulfonamides and trimethoprim, respectively) were determined. Analyses of tilapiafillet samples from fish exposed to sulfamethazine through feed (incurred samples) were conducted in order to evaluate themethod. .is new method was demonstrated to be fast, sensitive, and suitable for monitoring sulfonamides and trimethoprim intilapia fillet in health surveillance programs, as well as to be used in pharmacokinetics and residue depletion studies.

1. Introduction

Brazil is one of the five largest veterinarymarkets in the world,and aquaculture, in particular fish farming, is the fastestgrowing sector of animal food production in the country[1, 2]. In fish farming, antimicrobials, including sulfonamides,are used for the treatment of bacterial diseases. Sulfonamides(Figure 1) belong to an important group of synthetic anti-microbial agents that have been used in human and veterinarymedicine for over 60 years. Recently, these drugs have beenextensively employed in animals intended to produce food forhuman consumption since it is practically impossible to keep

the production environment free of pathogenic organisms.Sulfonamides have become a useful tool for achieving highlevels of productivity, thereby contributing to further growth,feed efficiency, and reduced mortality and morbidity [3].However, sulfonamide residues are a major concern becauseof their potential risk to human health by development ofbacterial resistance and adverse effects, such as allergic re-actions, in hypersensitive people [4].

Trimethoprim (Figure 1) is a diaminopyrimidine anti-microbial agent, which is active against a wide range ofGram-positive and Gram-negative microorganisms in-cluding Escherichia coli and some Klebsiella, Proteus,

HindawiJournal of Analytical Methods in ChemistryVolume 2018, Article ID 4506754, 10 pageshttps://doi.org/10.1155/2018/4506754

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and Staphylococcus species. In veterinarymedicine, it is oftenused in combination with a sulfonamide to increase theantimicrobial activity of the sulfonamides but is excretedfaster. Consequently, if no residues of sulfonamide aredetectable, no residues of trimethoprim would be expected.Trimethoprim is of low acute mammalian toxicity, and thereis no evidence for the potentiation of acute toxicity when it isadministered in combination with a sulfonamide [5].

At its 40th session, the Codex Alimentarius Commissionreported a maximum residue limit (MRL) value for sulfa-dimidine (sulfamethazine) of 100 µg·kg−1 in muscle, forspecies not specied [6]. According to the EuropeanCommission Regulation (EU) No. 37/2010 [7], for themuscle of n sh, the MRL value for individual sulfon-amides, or the combined total residues of all substancesbelonging to the sulfonamide group, is 100 µg·kg−1. In re-lation to trimethoprim, the MRL value is 50 µg·kg−1. eMRL value relates to the muscle and skin in natural pro-portions. In Brazil, the use of sulfonamides in farm-raised

sh is not permitted (it does not appear in the legislativeframework) and, therefore, its use is considered out of label(prohibited substance). However, for monitoring purposes(and taking actions), the Brazilian National Plan for Controlof Residues and Contaminants (PNCRC/Fish) establishesa reference limit of 100 μg·kg−1 for the residue of the in-dividual sulfonamides (sulfachlorpyridazine, sulfadoxine,sulfamerazine, sulfadiazine, sulfamethoxazole, sulfathiazole,sulfamethazine, sulfaquinoxaline, and sulfadimethoxine) orthe sum of them. Trimethoprim is not considered under thePNCRC/Fish sampling plan [8].

Studies on the determination of antimicrobial residues infoods of animal origin began in Belgium, the Netherlands,and Luxembourg in the late 1960s and early 1970s. In mostEuropean countries, research on residues and their appli-cation in inspection of slaughtered animals started later [9].In relation to the sample preparation step, strategies such assalting out liquid-liquid extraction [10], solid-liquid extrac-tion [11], and microscale matrix solid-phase dispersion [12]

NH

H2N

OCH3

OCH3CH3

OCH3

CH3

CH3

CH3

H2N

H2N

H2NH2N

NH2

NH

SO O

SO O

NH

NH

SO O

SO O

N

N

N

N

CH3

H2NSulfapyridine Sulfamerazine

Sulfathiazole

SulfamethoxazoleSulfamethazine

Sulfadimethoxine

Trimethoprim

Sulfamethoxypyridazine

NH

SO O

N

N

N

N

H2N

NH

SO O

H2N

NH

SO O

S

N

N

N

N

CH3O

OCH3

O

NO

Figure 1: Chemical structures of the sulfonamides and trimethoprim.

2 Journal of Analytical Methods in Chemistry

have been employed to perform the extraction and cleanupof sulfonamides from fish and other biological matrices.More recently, Ziarrusta et al. [13] used focused ultrasoundsolid-liquid extraction (FUSLE) for extraction of fluo-roquinolones from fish tissues. .e FUSLE method im-proves the extraction yield of target analytes (organiccompounds), quantitatively, from biota samples. Regard-ing the systems of separation and detection, the high per-formance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) is an analytical technique that has beenused in the determination of veterinary drug residues. In thisregard, a few sulfonamide multiresidue methods in foodmatrices have been described in the literature by this tech-nique [14, 15]. For instance, Abdallah et al. [16] determinedsulfonamide residues in sheep, pork, beef, chicken, anddromedary, Nebot et al. [17] in bovine milk, Tsai et al. [18] indifferent fish species, and Jansomboon et al. [19] in Pangasiuscatfish. Alternatively, a time-of-flight (TOF) mass spec-trometer provides high sensitivity and accurate massmeasurements (0.005Da), enabling the detection of lowconcentrations (ng·g−1) of residues and contaminants inhighly complex food matrices [15, 20]. Nevertheless, to ourknowledge, there is no reported multiresidue method for thecombined quantification of sulfonamides and trimethoprim intilapia fillet using liquid chromatography coupled to quad-rupole time-of-flight mass spectrometry (LC-QTOF/MS).

.e aim of this study was to develop and validate a rapid,simple (without the need of solid-phase extraction (SPE)cartridges or similar materials), and reliable multiresiduemethod for the identification and quantification of sulfon-amides and trimethoprim in tilapia fillets (Oreochromisniloticus) by LC-QTOF/MS, to be suitable for application inmonitoring programmes as well as in pharmacokinetic andresidue depletion studies. .e sample preparation involvedthe QuEChERS (Quick, Easy, Cheap, Effective, Rugged, andSafe) approach as described by Lehotay et al. [21]. .evalidation was conducted in-house based on the Commis-sion Decision 2002/657/EC [22] and Brazilian guideline[23]. To evaluate the precision of the method, analysis oftilapia fillet samples from fish exposed to sulfamethazinethrough feed (incurred samples) was also conducted.

2. Materials and Methods

2.1. Chemicals and Reagents. .e sulfonamide analyticalstandards (sulfathiazole (STZ), sulfamethoxazole (SMX),sulfamerazine (SMR), sulfamethoxypyridazine (SMPD),sulfadimethoxine (SDMX), sulfapyridine (SP), sulfametha-zine (SMZ)), and trimethoprim (TMP) were purchased fromSigma-Aldrich Company Ltd. (St. Louis, MO, USA). Allanalytical standards had a purity greater than 99.0%. Pri-mary secondary amine (PSA) was obtained from UnitedChemical Technologies, Inc. (UCT Inc., Bristol, PA, USA),and formic acid (98%) was purchased from Sigma-AldrichCompany Ltd. (St. Louis, MO, USA). Anhydrous magne-sium sulfate was supplied by J.T. Baker (Center Valley, PA,USA) and sodium acetate trihydrate from SpectrumChemical Mfg., Corp. (New Brunswick, NJ, USA). Methanol(MeOH) and acetonitrile (ACN) were obtained from

Honeywell Burdick & Jackson (Muskegon, MI, USA) andJ.T. Baker (Center Valley, PA, US), respectively. All solventswere of HPLC grade, and all reagents were of analyticalgrade. Ultra-pure deionized water was obtained from aMilli-Q Plus water purification system (Millipore, Bedford, MA,USA). Filtration of the aqueous mobile phase was performedusing polyvinylidene fluoride (PVDF) membranes, andpolytetrafluoroethylene (PTFE) membranes were used fororganic mobile-phase filtration, both with 0.22 µm pore sizeobtained from Millipore (Bedford, MA, USA).

2.2. Instrumentation. .e identification and quantitation ofsulfonamides and trimethoprim was carried out using anUPLC-Q-TOF system comprising an Acquity UPLC systemcoupled to a hybrid quadrupole orthogonal time-of-flight(Q-TOF) mass spectrometer (SYNAPTHDMSQ-TOFmassspectrometer) with electrospray source ionization (ESI) inpositive mode. .e software of acquisition control and datatreatment was the MassLynx version 4.1 (Waters Corp.,Milford, MA, USA). For sample preparation, the followingequipment were used: semianalytical balance (Tecnal;Boulder, CO, USA); analytical balance (Scientech, SA 210;Boulder, CO, USA); tubes stirring vortex type (IKA modelMS1 Minishaker, 2700 rpm; Wilmington, DE, USA); re-frigerated centrifuge (.ermo Scientific model HeraeusMultifuge 3 L-R; Madison, WI, USA); ultrasonic bath (Elmamodel Transsonic 660/H; Singen, Baden-Württemberg,Germany); Waring Commercial Blender, model 33BL79(New Hartford, CT, USA); and Ultra-Turrax IKA, model TP10N (Wilmington, DE, USA).

2.3. Solution Preparation. Standard stock solutions of SP,STZ, SMZ, SDMX, SMX, SMPD, SMR, and TMP wereprepared in acetonitrile at 1000 µg·mL−1, stored in 10mLbottles, and kept at −20°C. .ese solutions were used fora maximum period of 1 month. .e intermediate standardsolutions were prepared daily by dilution of stock solutionsin an appropriate buffer solution.

2.4. Blank and Incurred Fish Samples. .e blank samples oftilapia (Oreochromis niloticus) with no detectable analyteconcentration used for the development and validation ofthe analytical method were provided by a local producer (RioDoce fish farm, São João da Boa Vista, SP) with a guaranteethat the fish were not exposed to the compounds that werethe analytical focus of this work. Nonetheless, to ensure theviability of the blank samples, they were analysed, and thechromatograms did not show the presence of any in-terference at the retention time corresponding to the studiedanalytes. For validation of the analytical method, blanksamples and incurred samples (truly contaminated samples)were used, that is, samples of fish exposed to SMZ throughfeed, obtained from an experiment conducted at EmbrapaEnvironment, Jaguariuna, SP, Brazil, where tilapia weregiven SMZ at a dose level of 422mg·kg−1 body weight, for 11consecutive days. .e incurred samples used in this studywere from fish slaughtered by thermal shock and immersion

Journal of Analytical Methods in Chemistry 3

in an ice bath, 12 h after stopping medication. All sampleswere stored in a freezer (−20°C) until analysis [24]. eexperiment with sh to obtain the incurred samples wasapproved by the Ethics Committee on Animal Experimentsof Embrapa Environment (Protocol No. 001/2013) [25].

2.5. Sample Preparation byQuEChERS. Tilapia llet sampleswere ground using a domestic food processor. Trituratedsamples (2.5 g) were weighed in a 50mL polypropylene tube,and ACN (5mL) was added and then homogenized usinga Turrax for 30 seconds. e homogenized sample was thenadded of 5mL ACN, the tubes were shaken vigorously byvortexing for 1 min and placed in an ultrasonic bath for5min. Next, 2.0 g of anhydrous magnesium sulfate and0.75 g of sodium acetate were added to the homogenizedsamples and vortexed for 1 min and centrifuged at 17,500× gfor 10min, at 5°C. For sample cleanup, an aliquot of 5.0mL ofsupernatant was volumetrically pipetted to another tubecontaining 150mg of PSA and 0.5 g of anhydrous magne-sium sulfate. e tube was subsequently vortexed for 30seconds and centrifuged at 17,500× g again for 5min, at 5°C.A 2.0mL aliquot of the supernatant was pipetted andtransferred to another tube, and the solvent was completelyevaporated under nitrogen stream, in an ice bath, to avoidlosses of the analytes. Next, the residue was suspended in0.5mL of the mobile phase (ACN : 0.1% aqueous formicacid, 95 : 5 v/v). To facilitate the dissolution of analytes, thetubes were placed in ultrasonic bath for 5min. Finally, theresulting extracts were ltered through a cellulose lter unit(0.22 µm pore size) directly into the vial and injected in theLC-QTOF/MS system. A schematic representation of thesample preparation procedure is shown in Figure 2.

2.6. UPLC-QTOF/MS Conditions. e chromatographicseparation was performed on a reversed-phase analyticalcolumn Poroshell EC-120 C18 (50mm× 2.1mm, 2.7µm),supplied by Agilent Technologies (Santa Clara, CA, USA)preceded by a similar precolumn (30mm× 2.1mm, 2.7µm).e chromatographic separation was performed at 25°C. emobile phase was composed of (A) H2O : acetonitrile : formicacid (95 : 5 : 0.1%, v/v/v) and (B) H2O : acetonitrile : formicacid (5 : 95 : 0.1%, v/v/v), and the isocratic elution mode wasusedwith 70% (A) and 30% (B).e¢ow ratewas 0.2mL·min−1with a run time of 4min and injection volume of 5µL.

e following ionization conditions were established forthe ESI-QTOF/MS system: positive ionization mode, cap-illary voltage: 2.5 kV, detector voltage: 1.850 kV, sample conevoltage: 20.0 V, extraction cone voltage: 2.0 V, source tem-perature: 100°C, desolvation gas temperature: 300°C, nitro-gen gas ¢ow in the cone: 50 L·h−1, and desolvation ¢ow:400 L·h−1. e molecules of interest were quantied bymonitoring the signal related to the protonated molecularion m/z (M+H+). e sulfonamide and trimethoprimidentity was conrmed by obtaining the accurate mass of theprotonated molecular ion, as well as by the consideration offragment ions in order to obtain the identication points(IPs) according to Commission Decision 2002/657/EC [22](Table 1).

2.7. Validation Parameters. e purpose of this step was toestablish the performance parameters and the minimumrequirements of acceptance that must be satised such that theanalytical method presented in this study is considered val-idated. e recommendations of the European Community[22] and the Guide to Analytical Methods Validation of theBrazilian Ministry of Agriculture, Livestock, and Supply [23]were used as reference to perform the method validation.

After optimization of the preparation procedure (ex-traction and cleanup), the validation of the analyticalmethod was performed.e following validation parameterswere evaluated: selectivity; linearity, sensitivity, and matrixe¨ect; precision (intra- and interday); accuracy; and decisionlimit (CCα) and detection capability (CCβ). e limit ofdetection (LOD) and limit of quantication (LOQ) were alsoassessed to evaluate the potential use of the analyticalmethod in pharmacokinetic and residue depletion studieswhere lower LOD and LOQ are required. Selectivity of themethod was evaluated by comparing the chromatogramsobtained from blank samples (n � 10) and the samplesspiked with sulfonamides and trimethoprim standard so-lutions (n � 10). e chromatograms were evaluated for thepresence of the analytical signal at the same retention timeobserved for themass-to-charge ratio (m/z) of the analytes ofinterest.

2.5 ground sample+5 mL ACN

Turrax, 30 s

5 mL ACN

2.0 g anhydrous magnesium sulfate+0.75 sodium acetate

Vortex, 1 min

Centrifuge, 17,500g, 10 min, 5°C

5 mL supernatant+150 mg PSA+0.5 g anhydrous magnesium sulfate

Vortex, 30 s

Centrifuge, 17,500g, 10 min, 5°C

2 mL supernatant

Solvent evaporation in ice bath

Residue+0.5 mL mobile phase

Ultrasound, 5 mL

Filter in 0.22 µm membrane

UPLC-QTOF/MS

Figure 2: Schematic representation of the sample preparationprocedure.


Linearity was established from analytical curves obtainedby duplicate analysis of blank samples spiked with tri-methoprim and sulfonamides in the following concentra-tions: 5.0, 12.5, 25.0, 50.0, 75.0, 100.0, 125.0, and 250.0 ng·g−1..e results were analysed by the method of least squares ,and the linearity was expressed through the coefficient ofdetermination (R2) which was adopted as R2≥ 0.99, asrecommended by the Brazilian validation guide [23]. .ematrix effect was evaluated by comparing three differentconcentrations (12.5, 50.0, and 100.0 ng·g−1) of sulfonamidesand trimethoprim, prepared in solvent and fortified extracts..e evaluation was done by comparing the area of theanalytical signal in solvent with the area of analyte in thefortified extracts. Accuracy was evaluated by recovery tests ofthe spiked blank matrix at three concentration levels (10.0,20.0, and 40.0 ng·g−1) with five replicates of each spiked level,during 3 days. .e results were expressed as mean values(n � 15) of percentage of recoveries. .e coefficient ofvariation (CV%) is also reported.

.e precision of the method was determined in twosteps: intraday precision (repeatability) and interdays pre-cision (intermediate precision). Repeatability was expressedas the CV% of the results obtained with five replicates atthree different concentrations (10.0, 20.0, and 40.0 ng·g−1)analysed on the same day by the same analyst. .e in-termediate precision was expressed by CV% of the results ofthree different concentrations with five replicates of eachconcentration on three different days by the same analyst.

.e calculation of the decision limit (CCα) and thedetection capability (CCβ) was based on the CommissionDecision 2002/657/EC [22]. .e decision limit is defined asthe lowest concentration level at which the method candiscriminate with a statistical certainty of 1−α if the analyteis present. For substances with an MRL, the value of α isconsidered to be 5%. .e calculation was performed byanalysing 20 blank samples fortified with the analyte at theMRL level. .e concentration of the MRL plus 1.64 timesthe standard deviation corresponds to the CCα (α� 5%)..edetection capability (CCβ) is the lowest amount of thesubstance that can be detected, identified, and/or quantifiedin a sample with an acceptable error probability (β). Forsubstances with an MRL, the determination of CCβ can beaccomplished by the analysis of 20 blank samples fortifiedwith the analyte in the decision limit (CCα). .e value of

CCα plus 1, 64 times the standard deviation, corresponds tothe CCβ (β� 5%).

For each sulfonamide and trimethoprim, the LOD andLOQ were established by analysing the fortified matrix withstandard solution of the analytes. LOD was determinedbased on signal-to-noise approach. .us, LOD wasexpressed as the lowest concentration with a signal equal tothree times the signal-to-noise ratio. .e LOQ was taken asthe first level of the analytical curve, which was measuredwith acceptable precision (CV≤ 20%) [26].

3. Results and Discussion

.e representative sulfonamide veterinary drugs werechosen based on a study of their use in fish farming aroundthe world, those monitored by the Brazilian National Planfor Control of Residues and Contaminants (PNCRC/Fish) ofthe Brazilian Ministry of Agriculture, Livestock, and FoodSupply and those used for other animal species that couldpotentially be illegally employed in fish farming. .us,sulfamethazine, sulfathiazole, sulfadimethoxine, sulfamer-azine, sulfamethoxazole (monitored by the PNCRC/Fish[8]), sulfapyridine, sulfamethoxypyridazine, and tri-methoprim (regulated for veterinary use [7], although notregulated for use in fish farming in Brazil) were selected..emaximum residue limit (MRL) adopted for all the sulfon-amides (individual or the combined total residues) was100 μg·kg−1, and 50 μg·kg−1 for trimethoprim [7].

3.1. Sample Preparation Based on QuEChERS. Dispersivesolid-phase extraction (d-SPE) technique and QuEChERShave been previously used for the determination of veteri-nary drug residues in animal fluids and tissues [16, 27, 28],but not for the concomitant determination of sulfonamidesand trimethoprim in fish fillet. It is well known that the stepof sample preparation (extraction of analytes and cleanup ofthe extract) is crucial. .is approach can influence themagnitude of the matrix effect, depending on the amount ofendogenous substances from which it is coextracted. Ace-tonitrile has been widely used in the extraction of analytesfrom complex matrices as it extracts analytes with few in-terfering compounds (e.g., low amount of lipophilic coex-tractives from the sample) and further promotes theprecipitation of proteins. .is is necessary because the lower

Table 1: Elemental composition, retention time, the m/z experimental (precursors and fragment) ions, and mass error determined instandard solution for the studied analytes.

Compound MolecularformulaRetention time

(min)Monoisotopicmass (Da)

m/z experimental[M+H]+ (Da)

Mass error(ppm)

m/z experimentalfragment ion (Da)

Trimethoprim C14H18N4O3 0.83 290.1379 291.1460 1.0 123.0592Sulfapyridine C11H11N3O2S 1.08 249.0572 250.0650 0.0 156.0128Sulfamerazine C11H12N4O2S 1.18 264.0681 265.0760 0.4 108.0483Sulfathiazole C9H9N3O2S2 0.99 255.0136 256.0210 1.6 156.0127Sulfamethazine C12H14N4O2S 1.25 278.0837 279.0920 1.4 108.0475Sulfadimethoxine C12H14N4O4S 2.19 310.0736 311.0810 1.3 156.0771Sulfamethoxazole C10H11N3O3S 1.81 253.0521 254.0600 0.4 156.0124Sulfamethoxypyridazine C11H12N4O3S 1.44 280.0630 281.0710 0.7 156.0125


the quantity of interfering content present in the extract, theless matrix eëct is observed, which leads to a better qualityanalysis [29].

Kruve et al. [30] reported the minimizing matrix eëct inLC-ESI-MS analysis by using extrapolative dilution. It wasdemonstrated by several tests using QuEChERS samplepreparation procedure that the use of a greater volume ofacetonitrile for analyte extraction of complex matrices tendsto reduce the matrix eëct, possibly eliminating the matrixeëct if a suitable dilution is achieved. It should be men-tioned that although LC-ESI-QTOF/MS technique is veryselective, possible interference caused by matrix substancescan lead to suppression or an increase in the ionization of theanalytes of interest [31]. us, this study explores the ex-traction of sulfonamides and trimethoprim by usingQuEChERS procedure making use of acetonitrile as theextracting solvent and extrapolative dilution.

Preliminary studies have shown that for the quanti-cation of sulfonamides and trimethoprim in tilapia lletusing the proportion of acetonitrile : sample 4 : 1 (v/w)showed the best results with fewer coextracts, thus de-creasing the presence of interfering compounds. It isnoteworthy that although the amount of sample used in thisstudy was four times lower than that used by Lehotay et al.[21], it was possible to achieve an LOQ of 5 ng·g−1 for allanalytes, consequently to the LC-ESI-QTOF/MS systemused. Literature data show that the LOQ for SDZ was36 ng·g−1, using 5 g of the sample [32]. Stubbings and Big-wood [33] showed an LOQ for SP, STZ, SMZ, SDMX, SMX,and SMR of 50 ng·g−1, also using 5 g of the sample.

e addition of salts to promote the salting out eëct hasbeen shown to enhance the optimization of the analyterecovery percentages in multiresidue methods since it in-creases the solubility of these molecules in the organic phase[34, 35]. In the QuEChERS approach reported by Lehotayet al. [21], 6 g of anhydrous magnesium sulfate and 2.5 g ofsodium acetate trihydrate were used. In the present methodfor extracting sulfonamides and trimethoprim from tilapiallet, 2 g of anhydrous magnesium sulfate and 0.75 g ofsodium acetate trihydrate were employed. At the cleanupstep, PSA and/or C18 were used. Since no signicant var-iation was observed between them in relation to recoveryvalues, we opted for the use of PSA only.is nding may beobserved because the fat content in tilapia llet is low. ereare studies in matrices that have considerably higher fatcontent in which the concomitant use of PSA and C18 isrequired for a better cleanup of the sample extract [33].

3.2. Identity Conrmation of Analytes. On the basis ofCommission Decision 2002/657/EC [22], the identityconrmation of a substance is performed by a system ofidentication points (IPs). e mass accuracy of a high-resolution mass spectrometer acquires 2 IPs for the pre-cursor ion and 2.5 for each transition product. e resolutionof mass spectrometer used in this study (SYNAPT HDMSQ-TOF) is more than 10,000, which fall within the criteriaestablished by the guide as a high-resolution MS. Underthe conditions selected, the protonated molecule and one

fragmented ion for each analyte could be monitored, thusreaching the requirements to conrm their identity in ac-cordance with Commission Decision 2002/657/EC [22]. Forthe quantitative purpose, only the sulfonamides and trimeth-oprim molecular ions were monitored.

3.3. Analytical Method Validation. e method selectivitywas evaluated by analysing ten samples free of analytes(blank samples) and comparing them to the chromatogramsobtained from samples spiked with the sulfonamides andtrimethoprim. Peaks for interfering compounds with thesame retention times as the analytes of interest with the samem/z were not observed. erefore, the method performed issatisfactorily selective.

Figure 3 shows the chromatograms of each analytestudied.

100

(%)

(%)

(%)

(%)

(%)

(%)

(%)

(%)

0

Trimethoprim0.83

100

0

Sulfathiazole0.99

100

0

Sulfapyridine1.08

100

0

Sulfamerazine1.18

100

0

Sulfamethazine1.25

100

0

Sulfamethoxypyridazine1.44

100

0

Sulfamethoxazole1.81

100

01.0 2.0 3.0

Counts versus acquisition time (min)4.0 5.0

Sulfadimethoxine2.19

Figure 3: Extracted chromatograms of spiked samples with sul-fonamides and trimethoprim at concentration of 50.0 ng·g−1.


To study the linearity, sensitivity, and matrix effect, theanalytical results at the following concentrations werecompared: 5.0, 12.5, 25.0, 50.0, 75.0, 100.0, 125.0, and250.0 ng·mL−1. Measurements were carried out for theanalytes dissolved in the solvent, in the fortified extract, andin the fortified blank matrix (matrix-matched). .e matrixeffect, expressed as a percentage, was calculated from thedivision between the areas obtained for the analyte in solventand in the fortified extract, at the same concentration level[36]. .e highest matrix effect value observed was 18,98%,which is below the maximum acceptable value by the val-idation guides (20%) [22]. .us, the matrix effect wasconsidered irrelevant for this method. However, whencomparing the analytical curves in extract with the curves inthe fortified blank matrix, it was noted that the slope (an-gular coefficient) of the curve for thematrix-matched samplewas much lower, indicating the loss of analytes (sulfon-amides and trimethoprim) during sample preparation step(extraction and cleanup). .us, for the present methoda matrix-matched analytical curve must be employed.

Accuracy was evaluated from recovery tests (%), asrecommended by the Commission Decision 2002/657/ECwhen no certified reference material (CRM) is available [22]..e experiment was carried out through the recovery test ofthe spiked samples at 3 levels (10.0, 20.0, and 40.0 ng·g−1),evaluating each level using 5 independent replicates on 3consecutive days. Analytes SP, SMR, and TMP had satis-factory recovery values (between 79.5 and 103.6%), SMZ andSMPD showed intermediate recoveries (between 64.6 and80.0), and STZ, SDMX and SMX exhibit lower recoveryvalues (between 38.4 and 52.9) (Table 2). Low recoveryvalues for sulfonamides have been reported. Won et al. [37]reached a recovery of 58.8% for SDMX after extraction ofthis molecule from marine products, such as common eel,blue crab, shrimp, and flatfish, among others. Sulfonamides’low recoveries have also been reported in other matrices.Summa et al. [38] report recoveries for SMX and SDM,extracted from eggs, around 60% and 55%, respectively. Areview dealing about the presence of sulfonamides in edibletissues reports recoveries of various sulfonamides rangingfrom 40 to 67% for honey, 45–85% for pork veal, and57–63% for salmon muscle [39]. Although recovery valuesfound for some sulfonamides were below the percentageestablished in the validation guide [22], the method has beenshown to be precise (CV% found is within the value specifiedin the validation guide), and the required LOQwas achieved,which leads us to consider that the method is suitable for theintended purpose. Nevertheless, this corroborates the needto use matrix-matched analytical curves for the quantifi-cation of the analytes in samples of unknown origin.

.e precision of the method was determined throughintraday precision (repeatability) and interdays (in-termediate precision) at three spiked levels and wasexpressed as coefficient of variation (CV%). .e intradayand interdays precision were evaluated in the concentrationlevels at 10.0, 20.0, and 40.0 ng·g−1, with 5 replicates at eachlevel. Working in this concentration range, we can ensurethe precision and accuracy since in the most dispersivepoints, the CV is ≤20%. .e repeatability (analysed on the

same day and same equipment) and the interdays precision(intermediate precision) are shown in Table 3.

For compounds with concentration levels lower than100 ng·g−1, the Commission Decision 2002/657/EC [22] andBrazilian validation guideline [23] recommend a maximumacceptable CV≤ 20%. As shown in Table 3, the validationparameters (intraday and interdays precision) meet thespecifications recommended by both guides since theyrecommend a CV≤ 20%.

.e decision limit (CCα) is a parameter that takes intoaccount the precision of the method for establishing a criticalreference level, from which we can conclude that a sample isclassified as nonconforming with a probability of error of 5%.An additional critical parameter, detection capability (CCβ),is calculated for use with nonconforming samples in order toconfirm their concentration, and their identities are con-firmed with an error probability of 5% (β � 5%).

.e decision limit (CCα) and detection capability (CCβ)values for each of the analyte studied are shown in Table 4.For sulfonamides, values varied from 102.6 to 120.0 µg·kg−1and from 111.7 to 140.1 µg·kg−1 for CCα and CCβ, re-spectively. For trimethoprim, those values were 70.0 and89.9 µg·kg−1, respectively. .us, considering the MRL valuesof 100 µg·kg−1 and 50 µg·kg−1, respectively, for sulfonamidesand trimethoprim, established by the regulatory frameworkof the European Union in fin fish [7], we can conclude thatthe method reported here is suitable for application insurveillance programmes of residues of sulfonamides andtrimethoprim in fin fish muscle samples.

.e evaluation of LOD and LOQ for the determinationof sulfonamides and trimethoprim residues in tilapia filletwas performed using the matrix-matched analytical curvefortified with the analytes. .e LOD and LOQ of the methodwere 1.0 ng·g−1 and 5.0 ng·g−1 for all sulfonamides and tri-methoprim, respectively. .e LOQ was validated by analyseof 10 replicates that showed a CV≤ 20% for all of theanalytes. .is indicates that due to the low value of LOQobtained, the method can be used by restrictive regulatoryagencies of countries such as Japan [40], which, for themultiresidue method intended for quantification of veteri-nary drug residues in animal and fishery products, adopt forindividual sulfonamides and trimethoprim a LOQ value of10 ng·g−1 and 20 ng·g−1, respectively.

3.4. Analysis of Incurred Samples. To assess the methoddeveloped, analysis was performed on genuinely contami-nated (incurred) fish samples obtained from an experiment inlaboratory where the fishes were exposed to SMZ through thefeed. .is study was related to the effects of dietary exposureto SMZ on the haematological parameters and hepatic oxi-dative stress biomarkers in Nile tilapia [25]. .e residue ofSMZ in the muscle of 10 independent samples analysed in thesame day was 1,062.9± 53.2 ng·g−1 (mean value± standarddeviation), and the precision (CV%) was 5.0%. Due to thehigh concentration levels, the extract of the samples wasdiluted prior to injection to adjust the concentration to fit therange of the analytical curve. .is corroborates the precisionof the method and provides confidence that it is appropriate


for the intended purpose and can be used by regulatoryagencies in health surveillance programs, as well as inpharmacokinetics and residue depletion studies.

4. Conclusions

A multiresidue method for determination of sulfonamidesSTZ, SMX, SMR, SMPD, SDMX, SP, and SMZ and tri-methoprim (TMP) in tilapia fillet was developed and

validated. .e analytes selected were those most frequentlyused worldwide in fish farming and those with the greatestpotential for illegal use. QuEChERS approach with ex-trapolative dilution was shown to be a simple and in-expensive sample preparation process that can be easily usedin routine analysis. Quantitation by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS) showed to be a selective and low detectabilitymethod. .us, the method is suitable for application in

Table 3: Intraday and interdays precision of sulfonamides and trimethoprim.

Validation parametersSulfonamides and trimethoprim

SP STZ SMZ SDMX SMX SMPD SMR TMPIntraday precision (CV%)10 ng·g−1 12.8 11.7 11.6 7.7 6.4 7.1 11.3 8.320 ng·g−1 11.2 8.2 8.3 7.7 5.9 13.9 13.6 7.840 ng·g−1 12.9 14.9 15.0 12.3 7.9 6.1 14.5 10.6Interdays precision (CV%)10 ng·g−1 14.4 19.2 19.4 17.8 11.9 9.4 15.4 13.220 ng·g−1 19.3 17.2 11.8 3.4 4.2 9.0 16.7 17.740 ng·g−1 19.0 18.6 19.1 4.5 10.0 5.5 15.0 16.2SP, sulfapyridine; STZ, sulfathiazol; SMZ, sulfamethazine; SDMX, sulfadimethoxine; SMX, sulfamethoxazole; SMPD, sulfamethoxypyridazine;SMR, sulfamerazine; TMP, trimethoprim; CV, coefficient of variation.

Table 2: Validation parameters of sulfonamides and trimethoprim.

Validation parametersSulfonamides and trimethoprim

SP STZ SMZ SDMX SMX SMPD SMR TMPWorking range (ng·g−1) 5–250 5–250 5–250 5–250 5–250 5–250 5–250 5–250Linearity (R2) 0.9958 0.9914 0.9922 0.9992 0.9994 0.9935 0.9964 0.9984Sensibility 1174.07 633.811 2324.3 2670.56 1626.52 2057.08 1345.72 1588.92Matrix effect (%)12.5 ng·g−1 −18.98 −2.07 −11.19 −3.58 −5.22 9.57 −3.68 9.6550 ng·g−1 1.60 14.71 0.37 −3.28 1.13 −4.15 1.85 −0.12100 ng·g−1 0.13 −7.02 0.46 1.61 −1.77 3.74 0.79 −0.03Accuracy (% recovery (CV%))10 ng·g−1 83.9 (14.4) 52.9 (19.2) 69.1 (19.4) 49.6 (17.8) 45.4 (11.9) 80.0 (9.4) 79.5 (15.4) 92.0 (13.2)20 ng·g−1 91.5 (19.3) 38.4 (17.2) 72.4 (11.8) 47.4 (3.4) 41.8 (4.2) 66.8 (9.0) 85.4 (16.7) 88.2 (17.7)40 ng·g−1 103.6 (19.0) 41.3 (18.6) 68.0 (19.1) 51.1 (4.5) 43.5 (10.0) 64.6 (5.5) 81.2 (15.0) 91.5 (16.2)LOD (ng·g−1) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0LOQ (ng·g−1) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0SP, sulfapyridine; STZ, sulfathiazol; SMZ, sulfamethazine; SDMX, sulfadimethoxine; SMX, sulfamethoxazole; SMPD, sulfamethoxypyridazine;SMR, sulfamerazine; TMP, trimethoprim; LOD, limit of detection; LOQ, limit of quantitation; CV, coefficient of variation.

Table 4: CCα and CCβ values for sulfonamides and trimethoprim in tilapia fillet.

Validation parametersSulfonamidesa and trimethoprimb

SP STZ SMZ SDMX SMX SMPD SMR TMPLimit of decision (CCα), ng·g−1 119.8 110.9 114.0 102.6 102.9 105.9 120.0 70.0Detection capability (CCβ), ng·g−1 139.7 121.7 122.0 117.1 111.7 118.2 140.1 89.9SP, sulfapyridine; STZ, sulfathiazole; SMZ, sulfamethazine; SDMX, sulfadimethoxine; SMX, sulfamethoxazole; SMPD, sulfamethoxypyridazine; SMR,sulfamerazine; TMP, trimethoprim. a.e MRL value adopted for the calculation of CCα and CCβ for all sulfonamides was 100 ng·g−1 [6]. b.e MRL valueadopted for the calculation of CCα and CCβ for TMP was 50 ng·g−1 [6].


monitoring programmes of residues of sulfonamides andtrimethoprim in tilapia fillet, even by countries such as Japanthat adopt low LOQ values for analytical methods to be usedin food for determination of residues of substances such asveterinary drugs. Also, it was shown to be appropriate to beused in pharmacokinetic and residue depletion studies.

Conflicts of Interest

.e authors declare that there are no conflicts of interestregarding the publication of this paper.

Acknowledgments

.e authors gratefully acknowledge the financial supportreceived from São Paulo Research Foundation-AgilentTechnologies (FAPESP-Agilent, 2013/50452-5), the BrazilianCoordination for the Improvement of Higher EducationPersonnel (PROEX/CAPES, 3300301702P1), and the BrazilianNational Council of Technological and Scientific Develop-ment (CNPq). .e authors also thank Dr. Patricia Aparecidade Campos Braga for her technical assistance in reviewing themanuscript.

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